Effect of early endothelial function improvement on subclinical target organ damage in hypertensives

Endothelial dysfunction is acknowledged as a marker for subclinical target organ damage (STOD) in hypertension, though its therapeutic potential has not yet been clarified. This study assessed whether early endothelial function improvement (EEFI) reduced STOD in patients with essential hypertension (EH). We conducted a retrospective cohort analysis of 456 EH patients initially free from STOD. Endothelial function was assessed using brachial artery flow-mediated dilation (FMD), with values ≤ 7.1% indicating dysfunction. Patients were initially categorized by endothelial status (dysfunction: n = 180, normal: n = 276), and further divided into improved or unimproved groups based on changes within three months post-enrollment. During a median follow-up of 25 months, 177 patients developed STOD. The incidence of STOD was significantly higher in patients with initial dysfunction compared to those with normal function. Kaplan–Meier analysis indicated that the improved group had a lower cumulative incidence of STOD compared to the unimproved group (p < 0.05). Multivariable Cox regression confirmed EEFI as an independent protective factor against STOD in EH patients (p < 0.05), regardless of their baseline endothelial status, especially in those under 65 years old, non-smokers, and with low-density lipoprotein cholesterol levels ≤ 3.4 mmol/L. In conclusion, EEFI significantly reduces STOD incidence in EH patients, particularly in specific subgroups, emphasizing the need for early intervention in endothelial function to prevent STOD.

All patients received treatment as per the standards outlined in the Chinese guidelines for hypertension management 11 .Follow-up was conducted every 1 to 3 months by telephone appointment, encompassing both outpatient and inpatient visits.If patients did not comply, certain target organ assessments (e.g., CIMT) were postponed to a 6-month interval.The follow-up endpoint was the occurrence of STOD, with the interval from enrollment to STOD onset was defined as the survival period.The follow-up cut-off date was May 31, 2018, with a median follow-up time of 25 months (95%CI 10-63).Initially intended to follow 762 patients, the study ultimately included 456 due to exclusion criteria, losses to follow-up, and missing FMD data.Patients were categorized into groups based on initial endothelial function: impaired and normal.Within three months of enrollment, they were further subdivided into improved and unimproved groups based on improvement in endothelial function (Fig. 1).

Clinical data collection
Upon admission, data on participants' age, gender, drinking and smoking habits, duration of hypertension and medical history were collected.Current smokers were defined as individuals who smoked at least one cigarette daily for over six months.Drinkers were defined as males consuming over 25 g and females over 15 g of alcohol per day 10 .Participants were required to abstain from coffee and strong tea for half an hour before the examination.Following a 5-min rest, blood pressure and heart rate were measured with a HEM-7052 automatic sphygmomanometer (Omron, Japan), averaging three readings 10 .Height and weight were measured, and then body mass index (BMI) was calculated as weight (kg) divided by the square of height (m 2 ).Blood samples were drawn from the antecubital vein of all subjects in the morning after an 8-h fast.Subsequently, the samples were analyzed for total bilirubin, alanine aminotransferase, lactate dehydrogenase, uric acid, creatinine, total cholesterol (TC), high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol (LDL-C), and fasting serum glucose (FSG) using an ADVIA 2400 automatic analyzer (Siemens, Germany) 10 .Urinary albumin was assessed by immunoturbidimetry (Roche P800, USA), and urinary creatinine by colorimetric

Statistical analyses
Data processing and analysis utilized SPSS version 22.0.Tests for normal distribution and homogeneity of variance preceded analysis of continuous variables.Normally distributed data were reported as mean ± standard deviation, while non-normally distributed data were reported as median and interquartile distance.Categorical data were expressed as frequencies (percentages).Group differences were assessed using Pearson or Fisher's chi-square tests, Student's t-test, and Mann-Whitney U test.Kaplan-Meier survival curves and cumulative hazard curve for the occurrence of outcome events in the two groups of patients were plotted using the "survival + survminer" package in R software (version 4.2.1), with group differences compared using the logrank test.Finally, multivariate Cox regression analysis determined the effects of EEFI on STOD and identified additional potential risk factors.Subgroup analysis was performed, with results visualized using the "forestploter" package.Statistical significance was defined as a two-sided p-value < 0.05.

Comparison of patient baseline characteristics
This study included 456 patients with EH, 180 with endothelial dysfunction, and 276 with normal endothelial function.Eventually, 177 patients developed STOD.All patients included in the study were free from any previous cardiovascular diseases at baseline.Compared to those with normal function, the endothelial dysfunction group featured a higher proportion of men, smokers, and individuals with diabetes, and displayed increased age as well as extended duration of EH (p < 0.05).Additionally, this group had lower levels of FMD, but higher uric acid levels (p < 0.05).In terms of medication, patients with initial endothelial impairment used more statins and fewer β-blockers than those with normal function (p < 0.05).No significant differences were observed in other baseline data between the two groups (Table 1).
Patients were categorized based on initial endothelial function for further analysis.In the impaired group, the improved subgroup had significantly fewer smokers than the unimproved subgroup (p < 0.05), with similar other baseline characteristics.Despite similar blood pressure control, the improved subgroup used more reninangiotensin system (RAS) inhibitors and fewer diuretics than the unimproved subgroup (Table 2).In the normal group, those in the improved subgroup were younger with higher eGFR levels than the unimproved subgroup (p < 0.05).There were no significant abnormalities between the groups in terms of initial treatment medications and other baseline data (Table 3).

Relationship between EEFI and the incidence of STOD in EH patients
Kaplan-Meier survival curve analysis revealed a significantly higher incidence of STOD in EH patients with endothelial dysfunction than in those with normal function (Log-rank test, p < 0.001), as shown in Fig. S1 (Supplementary Fig. 1).Subsequent subgroup analysis with a cumulative hazard curve indicated that the group with improved endothelial function exhibited a significantly lower STOD incidence than the unimproved group (Log-rank test, p < 0.05) (Fig. 2).

Cox regression analysis of the incidence of STOD in EH patients
Multivariate Cox regression analysis was conducted with EEFI, factors with significant differences or trends in the univariate analysis, and traditional risk factors (age, gender, BMI, smoking, blood pressure, FSG, and LDL-C) as independent variables to examine their association with STOD incidence.In patients with initial endothelial impairment, age, female and LDL-C emerged as independent risk factors for STOD, whereas EEFI (HR = 0.53, 95% CI 0.35-0.79)served as a protective factor (Fig. 3a).Subgroup analysis showed that EEFI significantly reduced the risk of STOD in patients under 65, male, overweight, non-diabetic, non-smokers, with LDL-C below 3.4 mmol/L.Conversely, its impact was not significant in patients 65 or older, female gender, not overweight, diabetic, smokers, or with LDL-C at or above 3.4 mmol/L (Fig. 3b).As illustrated in Fig. 4a, for patients with normal endothelial function, EEFI (HR = 0.53, 95% CI 0.30-0.94)continued to act as a protective factor against STOD, whereas smoking was the sole independent risk factor.Notably, the subgroup analysis revealed similar results to those in patients with initially impaired endothelial function, except for gender, BMI, and diabetes, which were not yet statistically different, as shown in Fig. 4b.

Discussion
This retrospective cohort study analyzed 456 patients with EH over 16 years to evaluate the impact of EEFI on preventing STOD.Results showed that EH patients with endothelial dysfunction faced a significantly higher risk of developing STOD than those with normal function.More importantly, early improvement in endothelial function significantly lowered the cumulative incidence of STOD, particularly when blood pressure was maintained at target levels..In EH, endothelial dysfunction significantly increases the risk of developing STOD 18,19 .FMD is a well-established, accurate, and non-invasive indicator of peripheral endothelial function [6][7][8][9][10]13 and is widely utilized in clinical research. Sunet al. 19 found that adverse changes in FMD correlated with increases in LVH, UACR, and CIMT in a large cohort of EH patients.Furthermore, Yang et al. 20 in a prospective study involving 199 EH patients, demonstrated that FMD was independently linked to the occurrence of STOD.This cohort study reaffirmed that EH patients who initially had endothelial dysfunction were much more likely to develop STOD compared to those with normal function.Mechanistically, endothelial dysfunction impairs nitric oxide (NO) production, leading to reduced vasodilation and increased vascular resistance, contributing to the advancement of hypertension 4,17 . Aditionally, endothelial dysfunction is associated with increased oxidative stress and inflammation, further damaging vascular tissues and exacerbating blood pressure control 18 .Therefore, endothelial dysfunction is not only an independent predictor of STOD in EH patients but also a crucial target for clinical intervention.
This study showed that EEFI significantly reduced the cumulative incidence of STOD in EH patients, irrespective of their initial endothelial function.Studies have suggested that identifying and treating endothelial dysfunction can slow the progression of hypertension and decrease the risk of severe cardiovascular events 19,21 .Intervention studies have revealed that RAS inhibitors and statins not only provide foundational treatment but also enhance endothelial cell function, increase NO production, and improve vascular health [22][23][24] .After three months of consistent antihypertensive treatment, only a subset of patients experienced improvement in endothelial function, potentially due to the endothelial protective effects of RAS inhibitors and statins.Recent research has indicated that SIRT6, through epigenetic modulation, can prevent hypertension-induced endothelial Table 2. Clinical characteristics of patients with initial endothelial impairment who improved vs. those who did not improve after 3 months of enrollment treatment.Data are expressed as means ± SD or median (25th-75th).EH essential hypertension, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, TBil total bilirubin, ALT alanine aminotransferase, LDH lactate dehydrogenase, HDL-C high-density lipoprotein-cholesterol, LDL-C low-density lipoprotein-cholesterol, FSG fasting serum glucose, CIMT carotid intima-media thickness, UACR urinary albumin to creatinine ratio, LVMI left ventricular mass index, ACEI angiotensin-converting enzyme inhibitor, ARB angiotensin receptor blocker, FMD flow-mediated dilation.dysfunction 21 .Non-pharmacological interventions, such as regular aerobic exercise 25,26 , dietary changes (e.g., mediterranean diet 27 , omega-3 fatty acids 28 ), stress reduction techniques (e.g., yoga 29 ), and lifestyle modifications like smoking cessation 30 , have been shown to improve endothelial function and promote vascular health in recent studies.However, reports on the early enhancement of endothelial function to reduce STOD in EH patients remain scarce.This study enhances our understanding that EEFI can serve as a preventive strategy to lower the risk of STOD in hypertensives.This study also emphasized the importance of EEFI in specific populations.In hypertensive patients under 65 years, EEFI effects are more pronounced due to their greater vascular elasticity and stronger cellular regenerative capacity 31 .Male patients' positive response to EEFI may be due to their unique physiological and hormonal conditions.Research indicates that androgens may promote vasodilation in hypertensives by increasing the bioavailability of NO 32 .Overweight patients face a heightened risk of endothelial dysfunction, driven by elevated inflammation 33 .In this population, early intervention in endothelial function may reduce cardiovascular risks 34 .Hypertensive non-smokers typically exhibit lower oxidative stress 35 .Thus, they are better positioned to benefit from EEFI and show a stronger capacity for endothelial function recovery.Additionally, lower LDL-C and normal blood glucose support a healthy metabolic state, enhancing the endothelial cells' response to treatment and thereby promoting vascular health 36,37 .These findings underscore the need for targeted endothelial assessment and intervention in specific hypertensive subgroups to lower STOD risk and improve cardiovascular health, facilitating personalized treatment strategies.

Variables
However, this study had several limitations.The retrospective cohort design might have introduced information bias and missing variables, which were addressed through Cox regression analysis.Categorizing patients by endothelial function improvement within three months did not consider the effects of different medications or lifestyle changes.The single-center setting with a limited sample size, especially with few patients  having initial endothelial dysfunction, may constrain the broader applicability.Importantly, we did not analyze major adverse cardiovascular events, which are essential for understanding long-term cardiovascular risks.We also did not measure endothelial glycocalyx biomarkers, which could have provided deeper insights into endothelial health.Although FMD is the gold standard for assessing endothelial function, it is time-consuming, expensive, and requires specialized equipment and personnel.Alternative methods like reactive hyperemia index, pulse wave velocity, and serum biomarkers (e.g., endothelin-1 and C-reactive protein) could be more practical 38 .Lastly, our study included only Asian patients, limiting our findings' applicability to other racial and geographic populations.Future research should include a more diverse patient population to validate and broaden these findings.

Conclusion
In summary, this study demonstrated that meeting blood pressure targets in EH patients, combined with early enhancement of endothelial function, significantly decreased the risk of subsequent STOD.The results provide clinicians essential insights into assessing and intervening in endothelial function, recommending their incorporation into standard EH management protocols, particularly for patients under 65 years old, nonsmoking, and with LDL levels below 3.4 mmol/L.

Figure 2 .
Figure 2. Cumulative hazard curves for STOD in the improved (dotted line) and unimproved (solid line) groups of EH patients with initial impaired (a) and unimpaired (b) endothelial function.EH essential hypertension, STOD subclinical target organ damage.

Figure 3 .
Figure 3. Cox regression analysis of STOD in patients with essential hypertension who had initial impaired endothelial function (a) and its subgroups (b).STOD subclinical target organ damage, BMI body mass index, LDL-C low-density lipoprotein-cholesterol, EEFI early endothelial function improvement.

Figure 4 .
Figure 4. Cox regression analysis of STOD in patients with essential hypertension who had initial normal endothelial function (a) and its subgroups (b).STOD subclinical target organ damage, BMI body mass index, LDL-C low-density lipoprotein-cholesterol, eGFR estimated glomerular filtration rate, EEFI early endothelial function improvement.

Table 1 .
Clinical characteristics of hypertensives patients with and without initial endothelial impairment.

Table 3 .
Clinical characteristics of patients with initial normal endothelial function who improved vs. those who did not improve after 3 months of enrollment treatment.Data are expressed as means ± SD or median (25th-75th).EH essential hypertension, BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, TBil total bilirubin, ALT alanine aminotransferase, LDH lactate dehydrogenase, HDL-C high-density lipoprotein-cholesterol, LDL-C low-density lipoprotein-cholesterol, FSG fasting serum glucose, CIMT carotid intima-media thickness, UACR urinary albumin to creatinine ratio, LVMI left ventricular mass index, ACEI angiotensin-converting enzyme inhibitor, ARB angiotensin receptor blocker, FMD flow-mediated dilation.